Spatially Resolved FCS Detection for Faster Brightness Acquisition
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Solution Overview
Problem
Existing fluorescence correlation spectroscopy (FCS) methods, such as spot-variation FCS, require lengthy single measurements for each confocal volume, typically taking 10-100 seconds per measurement, which is inefficient and not optimal for minimizing exposure to excitation radiation, especially in biological samples.
Innovation Solution
A method utilizing a spatially resolved detector with individually readable detector elements and controlled adjustment of the excitation beam's extent to perform fluorescence correlation spectroscopy, allowing simultaneous acquisition of brightness information from multiple virtual apertures or pinholes, thereby increasing detection speed while minimizing sample exposure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a single confocal volume is measured using traditional FCS methods, then measurement precision is maintained, but measurement time increases significantly (10-100 seconds per volume)
Solution Approach 1:
The detector is divided into multiple independently readable detector elements arranged in groups around the optical axis. Each detector element can be read out individually, allowing simultaneous acquisition of brightness information from multiple virtual pinholes corresponding to different confocal volumes. This segmentation enables parallel measurement of multiple volumes instead of sequential measurement, dramatically reducing total measurement time while maintaining precision through individual element readout
Solution Approach 2:
The invention transitions from measuring a single confocal volume to simultaneously measuring multiple confocal volumes by utilizing the angular dimension. Detector elements are arranged at different angles around the optical axis, with each element detecting radiation from a different angular range. This angular dimensionality allows parallel acquisition of brightness information from multiple volumes without increasing temporal duration
2Productivity
If multiple confocal volumes are measured simultaneously using spatially resolved detector, then detection speed increases, but device complexity increases
Solution Approach 1:
A single spatially resolved detector performs multiple functions: it simultaneously detects radiation from multiple confocal volumes, replaces multiple individual detectors, and enables both sequential and parallel measurement modes. The detector elements can be individually read out or combined, providing versatile measurement capabilities without requiring multiple separate detection systems, thus increasing productivity while managing complexity
Solution Approach 2:
The invention creates virtual pinholes through software processing of detector element signals, replacing physical pinholes that would require separate mechanical components for each measurement volume. The detector elements are arranged to simulate multiple pinhole positions at different angles, allowing virtual aperture creation without additional physical apertures or complex mechanical switching mechanisms
3Adaptability or versatility
If traditional variable iris diaphragm is used to vary numerical aperture, then confocal volume size is adjusted, but measurement time increases due to sequential measurements
Solution Approach 1:
The system dynamically switches between different numerical apertures by selecting different detector elements or groups of elements for reading out. Each detector element corresponds to a different angular range and thus a different effective numerical aperture. This dynamic selection allows rapid switching between different confocal volume sizes without mechanical adjustment, enabling versatile measurement capabilities while maintaining fast detection speed
Solution Approach 2:
The invention replaces the mechanical variable iris diaphragm system with an electronic selection mechanism. Instead of physically adjusting aperture size with moving parts, the system uses software-controlled selection of detector elements to vary the effective numerical aperture. This substitution eliminates mechanical adjustment time and enables rapid switching between different confocal volume configurations
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly enhances the acquisition speed of brightness information, enabling simultaneous measurement from multiple virtual pinholes, thus reducing overall measurement time and minimizing sample exposure to excitation radiation.
Implementation Method 1
A first measurement block is implemented, each with at least one measurement process and a number of individual measurements. In a first measurement block, a Step A involves illuminating a sample with a focused beam of excitation radiation using an objective lens.
Implementation Method 2
The excitation radiation, such as laser radiation, can cause the emission of detection radiation by fluorophores that label structures, organelles, molecules, and/or regions of the sample.
Implementation Method 3
Step D comprises guiding the acquired detection radiation along a detection beam path and imaging the detection radiation onto a spatially resolved detector, which has a number of detector elements arranged in groups at equal intervals around an optical axis of the detection beam path and which can be read independently of one another.
Data Source
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AI summary
The invention relates to a method for acquiring brightness information of a specimen (48), in particular using fluorescence correlation spectroscopy. In a first measuring block, the specimen (48) is illuminated with a focussed beam of excitation radiation using a lens (47), wherein the excitation radiation is directed onto/into the specimen (48) at an angular range of a first numerical aperture. A first extent of the cross section of the beam is adjusted in the entrance pupil (EP) of the lens (47) for this purpose. A detection radiation is generated and detected in the specimen (48) in an excitation volume resulting from the illumination. The detected detection radiation is directed along a detection beam path (410) and mapped on a spatial-resolution detector (414) which comprises a number of detector elements (1 to 32) arranged at equal intervals about an optical axis (oA) of the detection beam path (410) which can be read independently of each other, and brightness information is acquired. Advantageously, the first numerical aperture is selected on the basis of the specimen (48) by acquiring values of at least a number of optical characteristics of the specimen (48) and the current first numerical aperture is determined and adjusted on the basis of the acquired values. The invention further relates to a device designed to carry out the method according to the invention.